The present invention relates to a novel protein involved in myocyte differentiation and DNA encoding the protein.
Genes, such as muscle creatine kinase, troponin, caveolin 3, xcex1-actin, and myosin, are reported to be expressed predominantly in the skeletal muscles. A family of transcription factors specifically expressed in the muscles, including myoD, myogenin, myf-5, and MRF-4/herculin/myf-6, have been cloned. These factors are phosphorylated nuclear proteins containing a helix-loop-helix (bHLH) motif, as required for both dimerization and DNA binding, and are believed to be determinants of the cell-specific differentiation program (Olson and Klein (1994), Genes and Dev. 8:1-8). When one of these factors is introduced into non-myogenic cells, differentiation into mature muscle cells is initiated (Weintraub et al. (1991), Science 251:761-766). The myoD family, a group of transcription factors, has been found to direct muscle formation, inhibit proliferation, activate differentiation and induce a contractile phenotype. While myoD and myf-5 are expressed within the proliferating myoblasts, myogenin and MRF-4 are not expressed until the myoblasts withdraw from the cell cycle in response to mitogen withdrawal. Based on these findings, it was demonstrated that myogenin and MRF-4 activate and maintain the expression of muscle-specific genes (Emerson (1993), Curr. Opin. Genet. Dev. 3:265-274), while myoD and myf-5 are thought to play a role in the proliferation of myoblasts. Other cell-cycle regulatory proteins, such as RB (Shiio et al. (1996), Oncogene 12:1837-1845, Wang et al. (1997), Cancer Research 57:351-354), p21 (Guo et al. (1995), Mol. Cell Biol. 15:3823-3829), cyclin D, cdk2, cdk4 (Kiess et al. (1995), Oncogene 10:159-166) and tumor suppressor gene p53 (Soddu et al. (1996), J. Cell Biol. 134:193-204) are involved in the muscle cell differentiation program. Recently, caveolin 3 (Song et al. (1996), J. Cell Biol. 271:15160-15165), xcex1-dystroglycan (Kostrominova and Tanzer (1995), J. Cell Biochem. 58:527-534) and DNA methyltransferases (Takagi et al. (1995), Eur. J. Biochem. 231:282-291) have been shown to play positive roles in myogenic differentiation.
An objective of the present invention is to provide a novel protein and gene involved in myocyte differentiation, and the production and use thereof.
The inventors carried out an antibody screening, using an antibody raised against a protein specific to immortalized cells, to isolate genes specifically expressed in the immortalized cells. Unexpectedly, a novel gene was isolated, which was not an initial objective gene. By analyzing the isolated gene, the inventors found that this gene is a novel gene showing no significant homology with any known genes deposited in the database, and is strongly expressed in skeletal muscle and undifferentiated cells. The inventors also analyzed the protein encoded by the gene, and found that the protein has an inhibitory effect on the differentiation of myoblasts into myotubes. The inventors also found that the protein interacts with p53, a transcription factor involved in tumor suppression, to inhibit the p53 transactivation function.
The present invention relates to a novel protein having an inhibitory effect on the differentiation of myoblasts into myotubes, and the gene encoding the protein, and the production and the use thereof. More specifically the present invention relates to:
(1) a protein comprising the amino acid sequence of SEQ ID NO:1, or a protein comprising said amino acid sequence in which one or more amino acids are substituted, deleted or added and exhibiting an inhibitory effect on the differentiation of myoblasts into myotubes;
(2) a protein encoded by DNA that hybridizes with the DNA comprising the nucleotide sequence of SEQ ID NO:2, wherein said protein exhibits an inhibitory effect on the differentiation of myoblasts into myotubes;
(3) a DNA encoding the protein according to (1);
(4) a DNA hybridizing with the DNA comprising the nucleotide sequence of SEQ ID NO:2, wherein said DNA encodes a protein exhibiting an inhibitory effect on the differentiation of myoblasts into myotubes;
(5) a vector containing the DNA according to (3);
(6) a transformant retaining the DNA according to (3) in an expressible manner;
(7) a method for producing the protein according to (1) or (2), said method comprising culturing the transformant according to (6);
(8) an antibody binding to the protein according to (1) or (2);
(9) a method of screening for a compound that binds to the protein according to (1) or (2), said method comprising the steps of:
a) contacting a test sample with said protein or a partial peptide thereof;
b) detecting the binding activity of the test sample to said protein or a partial peptide thereof; and
c) selecting a compound binding to said protein or a partial peptide thereof;
(10) a compound, binding to the protein according to (1) or (2), wherein said compound can be isolated using the method according to (9);
(11) a method of screening for a compound that promotes or inhibits the activity of the protein according to (1) or (2), the method comprising the steps of:
a) contacting myoblasts with said protein in the presence of a test sample;
b) detecting the differentiation of the cells into myotubes; and
c) selecting a compound which can increase or decrease the inhibitory activity of the protein, compared with its inhibitory activity as detected in the absence of said test sample;
(12) a method of screening for a compound that promotes or inhibits the activity of the protein according to (1) or (2), said method comprising the steps of:
a) providing p53-deficient cells with a vector expressing said protein, a vector expressing p53, and a vector expressing a reporter gene in response to p53;
b) contacting a test sample with said cells;
c) detecting the reporter activity in said cells; and
d) selecting a compound that can reduce or increase the reporter activity compared with the activity in the cells without contact with said test sample (control);
(13) a compound that promotes or inhibits the activity of the protein according to (1) or (2), wherein said compound can be isolated using the method according to (11) or (12); and
(14) a DNA comprising at least 15 nucleotides in length and specifically hybridizing with the DNA comprising the nucleotide sequence of SEQ ID NO:2.
The present invention relates to a novel protein, xe2x80x9cstriamin,xe2x80x9d that inhibits the differentiation of myoblasts into myotubes. (The inventors initially designated the protein xe2x80x9cstriatinxe2x80x9d in the original application (Japanese Patent Application No. Hei 10-115975), but another protein was later found to have the same name; hence the renaming to xe2x80x9cstriaminxe2x80x9d). The nucleotide sequence of striamin cDNA derived from mouse DNA is shown in SEQ ID NO:1, and the amino acid sequence of the protein encoded by the cDNA is shown in SEQ ID NO:2. As shown in SEQ ID NO:1, mouse striamin cDNA has an ORF encoding a protein of 149 amino acids. As determined by immunoprecipitation of the striamin protein translated in vitro (FIG. 2A), and by Western blotting of the recombinant striamin protein (FIG. 2B), the mouse-derived striamin protein has a molecular weight of about 18 kDa. Northern blot analysis showed that the striamin gene is expressed in the undifferentiated cells, and that the expression of this gene is inhibited during myoblast differentiation into myotubes (FIG. 4C). Overexpression of the gene actually blocked the differentiation of myoblasts into myotubes (FIG. 5). These facts suggest that the striamin protein is involved in the duration of the undifferentiated state of the cells.
The expression of the striamin protein also inhibited expression of the p53 transactivation function. The expression of this transcription factor is known to be upregulated during muscle differentiation (FIG. 7 and FIG. 8). The striamin protein was further shown to interact with p53 both in vivo and in vitro (FIG. 9 and FIG. 10). It is reported that p53 activity increases substantially in the process of muscle formation in vitro. Inhibition of p53 activity by striamin is quite consistent with the down-regulation of striamin during myogenesis. These facts suggest that striamin affects muscle formation through direct interaction with p53.
The striamin protein of the present invention may be prepared as a recombinant protein by making use of recombinant technology, and as a naturally occurring protein. For example, a recombinant protein can be prepared, as described below, by culturing cells transformed with DNA encoding the striamin protein. In addition, a naturally occurring protein can be isolated from tissues, such as skeletal muscles, using methods known by a person skilled in the art, for example, by performing affinity chromatography using an antibody that binds to the striamin protein as described below. The antibody may be a polyclonal or monoclonal antibody. Polyclonal antibodies can be prepared, for example, by obtaining serum from a small animal, such as a rabbit, that is immunized with the striamin protein, followed by purification using, for example, ammonium sulfate precipitation, protein A column, protein G column, DEAE ion exchange chromatography, and striamin protein coupled affinity column. Monoclonal antibodies can be prepared as follows. A small animal, such as a mouse, is immunized with the striamin protein. The mouse is then dissected to remove the spleen, which is subsequently homogenized to dissociated cells. These are then fused to mouse myeloma cells using a reagent such as polyethylene glycol, and the fused cells (hybridomas) thus obtained are subjected to the selection of clones producing antibodies against the protein. Subsequently, a hybridoma cell thus obtained is transplanted into a mouse intraperitoneally, and the ascites fluid is recovered from the mouse, followed by purification using, for example, ammonium sulfate precipitation, protein A column, protein G column, DEAE ion exchange chromatography, and striamin protein coupled affinity column.
When the antibody obtained is for human use (e.g., for antibody therapy), a humanized or human antibody is advantageous to reduce the immunogenicity. Among methods for humanizing antibodies, the CDR grafting method is well known. In this method, the antibody gene is cloned from the cell producing the monoclonal antibody and its antigen determinant portion is grafted to an existing human antibody. Alternatively, human antibodies can be prepared directly, by the same method used for conventional monoclonal antibodies, i.e., by immunizing a mouse whose immune system is replaced with a human""s immune system.
In addition, as well as preparing a native striamin protein, one skilled in the art can prepare modified proteins whose functions are equivalent to those of the native protein (e.g., an inhibitory effect on the differentiation of myoblasts into myotubes, binding activity to p53, an inhibitory effect on the p53 transactivation function), using a well-known method for modifying proteins, such as for substitution of amino acid residues in the protein. Spontaneous mutation of an amino acid in the protein may possibly occur. Thus, the proteins of the present invention include mutant proteins whose amino acid sequences differ from that of the native protein by amino acid substitution, deletion and/or addition, and whose function is equivalent to the native protein. The methods for modifying amino acids, which are well-known to one skilled in the art, include the site-directed mutagenesis system by PCR (GIBCO-BRL, Gaithersburg, Md.), the site-directed mutagenesis method using oligo-nucleotides (Kramer, W. and Fritz, H. J. (1987), Methods in Enzymol., 154:350-367), and Kunkel""s method (Methods in Enzymol. (1988), 85:2763-2766). Amino acid substitutions are made at typically 10 or less residues, preferably six or less residues and more preferably three or less residues.
A xe2x80x9cconservative amino acid substitutionxe2x80x9d is one in which an amino acid residue is replaced with another residue having a chemically similar side chain. Families of amino acid residues having similar side chains have been defined in the art. These families include amino acids with basic side chains (e.g., lysine, arginine, histidine), acidic side chains (e.g., aspartic acid, glutamic acid), uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine), nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan), beta-branched side chains (e.g., threonine, valine, isoleucine) and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine).
Any inhibitory effect of the proteins thus prepared on the differentiation of myoblasts into myotubes can be detected using, for example, a method such as that for determining differentiation potency by using cultured mouse C2C12 myoblast cell line. (When cultured in serum-free DMEM medium or DMEM medium containing 2% equine serum, mouse C2C12 myoblast cell line is differentiated into multinucleate myotube cells.) In this method, a C2C12 myoblast cell line is cultured in the presence of a test protein to determine the potency of the differentiation into multinucleate myotube cells (See Example 6). Binding of the prepared protein to p53 can be detected, for example, by contacting the two proteins in vitro or in vivo, which are then subjected to immunoprecipitation with an anti-p53 antibody, an antibody against the prepared protein or, if a tag is added to either protein, an antibody against the tag, and subsequently by Western blotting (see Examples 10 and 11). Inhibition of p53 transactivation by the prepared protein can be detected, for example, by determining the reporter activity of the cells into which both a vector expressing p53 and a vector carrying a p53-responsive reporter, and subsequently a vector expressing the prepared protein, are introduced. Reporter activity is then compared with that of a control, i.e., cells not harboring the vector expressing the prepared protein (See Examples 8 and 9).
It is also well within the art of a person with ordinary skill to obtain a protein functionally equivalent to the mouse striamin protein (SEQ ID NO:2) by isolating DNA showing significant homology with the DNA that encodes the mouse striamin protein (SEQ ID NO:1) or a part thereof, using technology such as a hybridization technique (Sambrook et al., Molecular Cloning 2nd ed. 9.47-9.58, Cold Spring Harbor Lab. Press, 1989). Thus, the proteins of the present invention also include those proteins that are encoded by DNA hybridizing with the DNA encoding the mouse striamin protein, and that are functionally equivalent to the mouse striamin protein (e.g., the protein that was detected in Example 5, which is encoded by a human transcript 3.1 kb in length). When hybridizing DNA is isolated from other organisms, animals including, but not limited to, humans, rats, rabbits, and cattle are used for the isolation. For this purpose, tissues such as skeletal muscles, in particular, are suitable. DNAs thus isolated, which encode proteins functionally equivalent to the mouse striamin protein, generally show significant homology with the DNA encoding the mouse striamin protein (SEQ ID NO: ). The term xe2x80x9csignificant homologyxe2x80x9d indicates a sequence identity of at least 40%, preferably at least 60%, more preferably at least 80%, and most preferably at least 95% at amino acid level. The degree of homology can be determined according to the algorithm described in the literature (Wilbur, W. J. and Lipman, D. J. Proc. Natl. Acad. Sci. USA (1983), 80:726-730).
The xe2x80x9cpercent identityxe2x80x9d of two amino acid sequences or of two nucleic acids is determined using the algorithm of Karlin and Altschul (Proc. Natl. Acad. Sci. USA 87:2264-2268, 1990), modified as in Karlin and Altschul (Proc. Natl. Acad. Sci. USA 90:5873-5877, 1993). Such an algorithm is incorporated into the NBLAST and XBLAST programs of Altschul et al. (J. Mol. Biol. 215:403-410, 1990). BLAST nucleotide searches are performed with the NBLAST program, score=100, wordlength=12. BLAST protein searches are performed with the XBLAST program, score=50, wordlength=3. Where gaps exist between two sequences, Gapped BLAST is utilized as described in Altschul et al. (Nucleic Acids Res. 25:3389-3402, 1997). When utilizing BLAST and Gapped BLAST programs, the default parameters of the respective programs (e.g., XBLAST and NBLAST) are used. The programs are available at the web site of the National Center for Biotechnology Information.
Examples of conditions used for the hybridization are as follows. For xe2x80x9clow stringencyxe2x80x9d hybridization, after prehybridization for at least 30 minutes at 55xc2x0 C. using xe2x80x9cExpressHyb Hybridization Solutionxe2x80x9d (CLONTECH), hybridization is carried out by adding a labeled probe and incubating for at least one hour at 37 to 55xc2x0 C., followed by washing the filter three times in 2xc3x97SSC containing 0.1% SDS for 20 minutes at room temperature and then once in 1xc3x97SSC containing 0.1% SDS for 20 minutes at 37xc2x0 C. For xe2x80x9cmedium stringencyxe2x80x9d hybridization, after prehybridization for at least 30 minutes at 60xc2x0 C. using xe2x80x9cExpressHyb Hybridization Solutionxe2x80x9d (CLONTECH), hybridization is carried out by adding a labeled probe and incubating for at least one hour at 60xc2x0 C., followed by washing the filter three times in 2xc3x97SSC containing 0.1% SDS for 20 minutes at room temperature and then twice in 1xc3x97SSC containing 0.1% SDS for 20 minutes at 50xc2x0 C. For xe2x80x9chigh stringencyxe2x80x9d hybridization, after prehybridization for at least 30 minutes at 68xc2x0 C. using xe2x80x9cExpressHyb Hybridization Solutionxe2x80x9d (CLONTECH), hybridization is carried out by adding a labeled probe and incubating for at least one hour at 68xc2x0 C., followed by washing the filter three times in 2xc3x97SSC containing 0.1% SDS for 20 minutes at room temperature and then twice in 0.1xc3x97SSC containing 0.1% SDS for 20 minutes at 50xc2x0 C.
The present invention also relates to the DNA encoding the striamin protein of the present invention. The DNA of the present invention may be any DNA including genomic DNA and synthetic DNA as well as cDNA, as long as it encodes the aforementioned striamin protein. The DNA of the present invention can be used, for example, to produce recombinant proteins. Such recombinant proteins can be prepared by inserting the DNA of the present invention (e.g., SEQ ID NO:1) into an appropriate expression vector. This is then introduced into appropriate cells to obtain a transformant, followed by culturing the transformants, and by purifying the expressed protein. Cells used for the production of recombinant proteins include, but are not limited to, mammalian cells such as COS, CHO, and NIH3T3 cells, insect cells such as Sf9 cells, yeast cells, and E. coli cells. Although vectors used for expression of recombinant proteins in the cell will vary depending on the host cells, such vectors include, for example, pcDNA3 (Invitrogen) and pEF-BOS (Nucleic Acids Res. 1990, 18 (17), p. 5322) for expression in mammalian cells, xe2x80x9cBAC-to-BAC baculovirus expression systemxe2x80x9d (GIBCO BRL) for expression in insect cells, xe2x80x9cPichia Expression Kitxe2x80x9d (Invitrogen) for expression in yeast cells, and pGEX-5X-1 (Pharmacia) and xe2x80x9cQIAexpress systemxe2x80x9d (Qiagen) for expression in E. coli cells. Introduction of the vector into the host cells can be carried out using methods including the calcium phosphate, DEAE dextran, cationic liposome DOTAP (Boehringer Mannheim), electroporation, and calcium chloride methods. The transformants can be cultured using a method well known to one skilled in the art, using an appropriate existing method, depending on the properties of the particular transformant. Recombinant proteins may be purified from the transformants thus obtained may be carried out using, for example, the method described in the literature xe2x80x9cThe Qiaexpressionist Handbook, Qiagen, Hilden, Germany.xe2x80x9d
The DNA of the present invention can be used in gene therapy for diseases caused by mutations that have occurred in the DNA. As used in gene therapy, the DNA of the present invention is inserted into a vector such as adenoviral (e.g., pAdexLcw) or retroviral (e.g., pZIPneo) vectors for in vivo administration. Administration can be carried out via either an ex vivo or in vivo process.
The present invention also features a DNA molecule that contains at least 15 nucleotides, and that can specifically hybridize with the DNA encoding the striamin protein of the present invention or the complementary DNA thereof. The term xe2x80x9cspecifically hybridizexe2x80x9d indicates that no significant cross-hybridization occurs to DNA encoding other proteins under standard hybridization conditions, preferably under stringent hybridization conditions. Such DNA molecules include probes, primers and nucleotides, and nucleotide derivatives (e.g., antisense oligonucleotides ribozymes, etc.), that can specifically hybridize to the DNA encoding the protein of the present invention or to the complementary DNA thereof.
An xe2x80x9cisolated nucleic acidxe2x80x9d is a nucleic acid the structure of which is not identical to that of any naturally occurring nucleic acid or to that of any fragment of a naturally occurring genomic nucleic acid spanning more than three separate genes. The term therefore covers, for example, (a) a DNA which has the sequence of part of a naturally occurring genomic DNA molecule but is not flanked by both of the coding sequences that flank that part of the molecule in the genome of the organism in which it naturally occurs; (b) a nucleic acid incorporated into a vector or into the genomic DNA of a prokaryote or eukaryote in a manner such that the resulting molecule is not identical to any naturally occurring vector or genomic DNA; (c) a separate molecule such as a cDNA, a genomic fragment, a fragment produced by polymerase chain reaction (PCR), or a restriction fragment; and (d) a recombinant nucleotide sequence that is part of a hybrid gene, i.e., a gene encoding a fusion protein. Specifically excluded from this definition are nucleic acids present in mixtures of different (i) DNA molecules, (ii) transfected cells, or (iii) cell clones: e.g., as these occur in a DNA library such as a cDNA or genomic DNA library.
The term xe2x80x9csubstantially purexe2x80x9d as used herein in reference to a given polypeptide means that the polypeptide is substantially free from other biological macromolecules. The substantially pure polypeptide is at least 75% (e.g., at least 80, 85, 95, or 99%) pure by dry weight. Purity can be measured by any appropriate standard method, for example, by column chromatography, polyacrylamide gel electrophoresis, or HPLC analysis.
The present invention includes an antisense oligonucleotide hybridizing to any portion of the nucleotide sequence of, for example, SEQ ID NO:2. Preferably, such an oligonucleotide is antisense to a continuous 15 nucleotides or more in length in the nucleotide sequence of SEQ ID NO:2. More preferably, the aforementioned continuous sequence 15 nucleotides or more in length contains a translation initiation codon.
Derivatives or modified oligonucleotides can be used as an antisense oligonucleotide. Such modified nucleotides include lower alkylphosphonate-modified, such as methylphosphonate- or ethylphosphonate-modified, phosphorothioate-modified, and phosphoroamidate-modified nucleotides.
As used here, the term xe2x80x9cantisense oligonucleotidesxe2x80x9d means not only an oligonucleotide complementary to all of the continuous nucleotides comprising the given region of DNA or mRNA, but also oligonucleotides having one or more nucleotides mismatched against the continuous nucleotides, as long as DNA or mRNA and the oligonucleotides are able to specifically hybridize to the nucleotide sequence of SEQ ID NO:2.
Such DNAs include continuous nucleotide sequences at least 15 nucleotides in length, showing at least 70%, preferably at least 80%, more preferably at least 90%, and most preferably at least 95% homology with the nucleotide sequence of SEQ ID NO:2. An algorithm that can be used to determine the extent of homology is given herein. These DNAs are useful as probes to detect or isolate the DNA encoding the protein of the present invention, according to the methods described below in the Examples. They are also useful primers for amplification.
The antisense oligonucleotide derivatives of the present invention act upon the cells producing the protein of the present invention. They bind to the DNA or mRNA encoding the protein, inhibiting the transcription or translation of the protein and promoting the degradation of the mRNA. As expression of the protein is inhibited, there is an inhibitory effect on the functioning of the protein.
The antisense oligonucleotide derivatives can be formulated into external preparations such as liniments and poultices by mixing with a suitable base material, which is inert to the derivatives.
If necessary, the derivatives can be formulated into tablets, powders, granules, capsules, liposome capsules, injections, solutions, nose-drops and into freeze-dried agents, by adding excipients, isotonic agents, dissolving auxiliaries, stabilizers, preservatives and pain-killers. These formulations can be prepared using a standard technique.
An antisense oligonucleotide derivative of the present invention could be given to a patient by direct application onto the affected site or by intravascular administration. A mounting medium for including antisense derivatives can also be used to increase sustainability and membrane-permeability of the formulations. For example, liposome, poly-L lysine, lipid, cholesterol and lipofectin or derivatives thereof can be used.
A range of dosages of the antisense oligonucleotide derivatives, from 0.1 to 100 mg/kg, can be administered, depending on the patients"" conditions.
The antisense oligonucleotides, or an inhibitor containing an antisense oligonucleotide, inhibit the expression of the protein of the present invention, and are thus useful in inhibiting its biological activity.
The present invention also features a screening method for a compound that binds to the protein. The screening method comprises the following steps of:
(a) contacting a test sample with the protein of the present invention or a partial peptide thereof;
(b) detecting the binding activity of the test sample to the protein of the present invention or a partial peptide thereof; and
(c) selecting a compound binding to the protein of the present invention or a partial peptide thereof.
The protein of the present invention used for screening may be recombinant or naturally occurring protein, or may be a partial peptide thereof. Any test sample can be used without particular restriction, including, for example, cell extracts, culture supernatants, products from fermented microorganisms, extracts from marine organisms, plant extracts, purified or crude proteins, peptides, nonpeptidic compounds, synthetic low molecular weight compounds and natural compounds.
A number of methods well-known to one skilled in the art can be used to screen for a protein binding to the protein of the present invention utilizing the protein of the present invention. One of these screening methods is immunoprecipitation. Typically, immunoprecipitation is conducted as follows. The gene encoding the protein of the present invention is inserted downstream of a promoter provided for expressing foreign genes, such as pSV2neo, pcDNA 1, and pCD8, to express the gene, for example, in mammalian cells. Any commonly available promoter may be used for the expression, including SV 40 early promoter (Rigby in Williamson (ed.), Genetic Engineering Vol.3, Academic Press, London, p. 83-141 (1982)), EF-1xcex1 promoter (Kim et al., Gene, 91:217-223 (1990)), CAG promoter (Niwa et al., Gene, 108:193-200 (1991)), RSV LTR promoter (Cullen, Methods in Enzymology, 152:684-704 (1987)), SR xcex1 promoter (Takebe et al., Mol. Cell. Biol. 8:466 (1988)), CMV immediate early promoter (Seed and Aruffo, Proc. Natl. Acad. Sci. USA, 84: 3365-3369 (1987)), SV 40 late promoter (Gheysen and Fiers, J. Mol. Appl. Genet., 1:385-394 (1982)), and Adenovirus late promoter (Kaufman et al., Mol. Cell. Biol., 9:946 (1989), HSV TK promoter.
Any method for introducing and expressing a foreign gene in animal cells may be used to express the gene, including electroporation (Chu, G. et al., Nucl. Acid. Res. 15:1311-1326 (1987)), a calcium phosphate method (Chen, C. and Okayama, H. Mol. Cell. Biol. 7:2745-2752 (1987)), a DEAE dextran method (Lopata, M. A. et al., Nucl. Acid. Res. 12: 5707-5717 (1984); Sussman, D. J. and Milman, G., Mol. Cell. Biol., 4:1642-1643 (1985)), Lipofectin method (Derijard, B., Cell, 7:1025-1037 (1994); Lamb, B. T. et al., Nature Genetics, 5:22-30 (1993); Rabindran, S. K. et al., Science, 259:230-234 (1993)).
The protein of the present invention can be expressed as a fusion protein containing a monoclonal antibody recognition site, the specificity of which has been defined by introducing the monoclonal antibody recognition site (epitope) at the N or C terminus of the protein of the present invention. Epitope-antibody systems used for this purpose are commercially available (Jikken Igaku, Experimental Medicine, 13:85-90 (1995)). A number of vectors provided for expression of a gene as a fusion protein fused with xcex2-galactosidase, a maltose binding protein, glutathione S-transferase, or a green fluorescent protein (GFP) via a multi-cloning site available from commercial sources.
To limit variation in characteristics between the protein of the present invention and its fusion protein, an epitope of restricted size is introduced to prepare the fusion protein. This can range from just below to just above ten amino acids. Such methods have been reported. A combination of an epitope, such as polyhistidine (His-tag), influenza hemagglutinin HA, human c-myc, FLAG, Vesicular stomatitis viral glycoprotein (VSV-GP), T7 gene 10 protein (T7-tag), human herpes simplex viral glycoprotein (HSV-tag) or E-tag (epitopes on monoclonal phages), and a monoclonal antibody that recognizes the epitope can be used as an epitope-antibody system to screen for a protein binding to the protein of the present invention (Jikken Igaku (1995), Experimental Medicine, 13:85-90).
In immunoprecipitation, an immunocomplex is formed when these antibodies are added to the cell lysate prepared using appropriate detergent. The immunocomplex comprises the protein of the present invention, a protein capable of binding to it, and the antibody. In addition to using the antibody against the epitope mentioned above, an antibody raised against the protein of the present invention can be used for the immunoprecipitation. Antibodies against the protein of the present invention can be prepared, for example, by introducing the gene encoding the protein of the present invention into an appropriate E. coli expression vector to express the protein in E. coli cells, purifying the expressed product, and immunizing animals, such as rabbits, mice, goats, and chickens, with the purified protein. Alternatively, the antibodies against the protein of the present invention can be prepared by immunizing the above animals with synthetic partial peptides of the protein of the present invention.
Immunocomplexes can be precipitated, for example, by using Protein A Sepharose or Protein G Sepharose, if the antibody is a mouse IgG antibody. When the protein of the present invention is prepared as fusion proteins fused with epitopes such as GST, substances specifically binding to the epitopes, such as glutathione-Sepharose 4B, can be used to form the immunocomplexes in the way that an antibody against the protein of the present invention is used.
A standard method of immunoprecipitation may be carried out as described in the literature; for example, Harlow, E and Lane, D: Antibodies, pp. 511-552, Cold Spring Harbor Laboratory publications, New York (1988).
SDS-PAGE is typically used to analyze the immunoprecipitated proteins. By using a gel with appropriate density, the bound proteins can be resolved by molecular size. In this procedure, the cells are cultured in a medium containing a radioisotope, such as 35S-methionine or 35S-cysteine, to label the proteins in the cells. The detection sensitivity is thereby increased, since, in general, proteins bound to the protein of the present invention are difficult to detect using conventional protein-staining methods such as Coomassie and silver staining. Once the molecular size of the protein is clarified, the protein of interest can be directly purified from SDS-polyacrylamide gel and subjected to sequencing.
West-Western blotting (Skolnik, E. Y. et al. Cell (1991), 65:83-90), for example, may be used to isolate proteins binding to the protein of the present invention by using the protein. In this method, the isolation is carried out by constructing a cDNA library from cells, tissue or an organ presumed to express binding proteins of the protein of the present invention (for example, myoblast cells and NIH3T3 cells) using a phage vector (e.g., xcexgt11, ZAP), expressing the vectors on LB-agarose, fixing the expressed proteins on the filter, reacting the filter with the labeled and purified protein of the present invention, and detecting the plaques expressing the proteins bound to the protein of the present invention through the label. The methods of labeling the protein of the present invention include those utilizing the affinity between biotin and avidin; those using an antibody binding specifically to the protein of the present invention, or a peptide or polypeptide fused to the protein of the present invention (for example, GST); those using a radioisotope; and those using fluorescence.
Another screening method of the present invention is to use the two-hybrid system using cells (Fields, S., and Sternglanz, R., Trends. Genet. (1994), 10:286-292). In two-hybrid systems methods, such as xe2x80x9cMATCHMAKER Two-hybrid System,xe2x80x9d xe2x80x9cMammalian MATCHMAKER Two-hybrid Assay Kit,xe2x80x9d and xe2x80x9cMATCHMAKER One-Hybrid Systemxe2x80x9d (Clontech), or xe2x80x9cHybriZAP Two-Hybrid Vector Systemxe2x80x9d (Stratagene), and also as described in xe2x80x98Characterization of SAP-1, a protein recruited by serum response factor to the c-fos serum response elementxe2x80x99 (Dalton S and Treisman R (1992), Cell 68:597-612), the protein of the present invention is fused to the SRF binding region or GAL4 binding region and expressed in yeast cells.
A cDNA library was prepared from cells predicted to express a protein that will bind to the protein of the present invention so as to express proteins fused to the VP16 or GAL4 transcriptional activation region. The cDNA library is then introduced into the above yeast cells, and the cDNA derived from the library is isolated from the positive clones detected (when a protein binding to the protein of the present invention is expressed in yeast cells, the binding of the two activates a reporter gene making positive clones detectable). The isolated cDNA can be introduced into E. coli to express a protein encoded by it. This method also allows preparation of a protein binding to the protein of the present invention and the gene encoding it. The reporter genes include Ade2, LacZ, CAT, and luciferase genes as well as HIS3 gene.
Affinity chromatography can be used to screen for the compound binding to the protein of the present invention. For instance, the protein of the present invention is immobilized on the support of the affinity column, to which a test sample is applied. The test sample is selected to express the protein that binds to the protein of the present invention; a cell extract or cell lysate can be used. After the test sample is applied, the column may be washed to prepare the protein bound to the protein of the present invention.
The amino acid sequence of the protein thus obtained may be analyzed and, based on the result, oligo-DNA is synthesized and used as a probe for screening the cDNA library to obtain the DNA encoding the protein.
The present invention may include use of a biosensor in which a surface plasmon resonance phenomenon is utilized to detect or determine the bound compounds. The biosensors utilizing a surface plasmon resonance phenomenon (e.g., BIAcore, Pharmacia) enable the real-time observation of the interaction between the protein of the present invention and the test compound as surface plasmon resonance signals, using a small amount of the protein without the need for labeling. Consequently, the binding of the protein of the present invention and the test compound can be estimated using a biosensor such as BIAcore.
Among methods for isolating compounds, not limited to proteins, that bind to the protein of the present invention, those methods of screening for molecules that bind to the protein of the present invention by making synthetic compounds, a natural substance bank, or a random phage peptide display library, act on the immobilized protein of the invention. Furthermore, methods of screening using a high-throughput based on combinatorial chemistry techniques are well known to one skilled in the art. (Wrighton N C; Farrell F X; Chang R; Kashyap A K; Barbone F P; Mulcahy L S; Johnson D L; Barrett R W; Jolliffe L K; Dower W J. xe2x80x98Small peptides as potent mimetics of the protein hormone erythropoietinxe2x80x99, Science (United States) Jul. 26, 1996 273:458-64, Verdine G L., xe2x80x98The combinatorial chemistry of naturexe2x80x99. Nature (England) Nov. 7, 1996, 384:11-13, Hogan J C Jr., xe2x80x98Directed combinatorial chemistryxe2x80x99. Nature (England) Nov. 7, 1996, 384:17-9).
The present invention also relates to a method for screening for a compound able to promote or inhibit the activity of the protein of the invention. Since the protein of the present invention has inhibitory activity on the differentiation of myoblasts into myotubes, a compound able to promote or inhibit activity of the protein of the invention can be screened by using this activity as an indicator. Such screening can be done using a method comprising the steps of:
(a) exposing the protein of the present invention to myoblast cells in the presence of a test sample,
(b) detecting the differentiation of the cells into myotube cells, and
(c) selecting a compound that increases or reduces the inhibitory effect of the protein of the present invention by comparing with the results of the assay performed in the absence of the test sample.
The protein of the present invention used for the screening can be a naturally occurring protein or a recombinant protein, or a purified protein or the supernatant of cell culture (when the protein is secreted from the cell).
There are no particular restrictions as to the test samples used. For example, cell extracts, culture supernatants, products from fermented microorganisms, extracts from marine organisms, plant extracts, purified or crude proteins, peptides, nonpeptidic compounds, synthetic low molecular weight compounds and natural compounds may be used. Alternatively, a compound obtained by the aforementioned screening for compounds binding to the protein of the present invention can be used as a test sample.
Myoblasts used for detecting the differentiation into myotube cells, preferably include, but are not limited to, C2C12 myoblasts. The differentiation into myotubes can be detected by a method, such as that for determining the differentiation potency of the C2C12 myoblast cell line into multinucleate myotube cells when they are cultured in the presence of both the test sample and the protein of the present invention, using the culture system of the mouse C2C12 myoblast line described in the Examples (the mouse myoblast cell line C2C12 differentiates into multinucleate myotube cells when cultured in the DMEM medium free of serum or containing 2% equine serum).
The protein of the present invention has here been shown to inhibit the transcriptional factor activity of p53. Therefore, a compound promoting or inhibiting the activity of the protein can also be screened conducted using the transcriptional factor activity as an indicator. This screening can be carried out by:
(a) providing p53-deficient cells in which vectors expressing the protein, p53 and a reporter gene responsive to p53 have been introduced,
(b) exposing the test sample to the cells,
(c) detecting the reporter activity in the cells, and
(d) selecting a compound that increases or reduces the reporter activity compared with the activity detected in the absence of the test sample (control).
Specifically, a test sample is added to the detection system described in Examples 8 and 9, in which inhibition of the transcriptional factor activity of p53 by striamin is detected, reporter activity is detected, and then the compound altering the activity may be selected. There are no particular restrictions as to the test samples used. For example, cell extracts, culture supernatants, products from fermented microorganisms, extracts from marine organisms, plant extracts, purified or crude proteins, peptides, nonpeptidic compounds, synthetic low molecular weight compounds, and natural compounds may be used. Alternatively, a compound obtained by the aforementioned screening for compounds binding to the protein of the present invention can be used as a test sample.
The striamin expression vector may express striamin fully or it may express a partial peptide of striamin which can inhibit the transcriptional factor activity of p53 (e.g., C-terminus of striamin). Preferably, the chosen p53 vector should express p53 controllably. Such vectors include temperature-sensitive p53 expression vectors.
A plasmid expressing the reporter in response to p53 has the reporter gene located downstream of a p53 responsive sequence. xe2x80x9cATGCTTGCCCxe2x80x9d (SEQ ID NO:17) may be used as the p53 responsive sequence. There are no particular restrictions as to the reporter gene used as long as it has a detectable response. Genes such as those for luciferase and xcex2-galactosidase can be used.
Preferably, p53-deficient cells are used in vector introduction to avoid the expression of the reporter gene by the action of endogenous p53. Such cells include p53-/-murine fetal fibroblast cells.
If a reduction in reporter activity is detected during screening, compared with the activity found in the absence of the test sample (control), then the test sample used is determined as being a compound (or containing a compound) that promotes the activity of the protein of the present invention. Likewise, when the test sample increases the reporter activity, it is determined as being a compound (or containing a compound) that inhibits the activity of the protein of the present invention.
As used in the screening method, xe2x80x9ca compound that promotes or inhibits the activity of the protein of the present inventionxe2x80x9d is any compound, without limit, promoting or inhibiting the signal transduction via the protein of the present invention. Specifically, such a compound is not limited to ones that promote or inhibit the activity by directly acting on the protein of the present invention. A compound that promotes or inhibits the signal transduction by action on any of the downstream factors from the protein of the present invention in the signal transduction is also included.
The compounds obtained from the screening of the present invention will be candidate agents that promote or inhibit the activity of the protein of the present invention, or that promote or inhibit the signal transduction via the protein of the present invention, for a disease associated with the protein of the present invention. The compounds obtained using the screening method of the present invention include any substance in which a portion of the structure of a compound having the binding activity to the protein of the present invention that is obtained using the screening method of the present invention and has been altered by addition, deletion and/or substitution.
To use the compounds obtained with the screening method of the present invention as drugs for humans and other mammals such as mice, rats, guinea pigs, rabbits, chickens, cats, dogs, sheep, pigs, cattle, monkeys, baboons, and chimpanzees, the compounds may be formulated for administration to the patients by a well-known pharmaceutical preparation method, as well as direct administration of the isolated compounds. For example, the drugs can be administered orally in the form of sugar-coated tablets, capsules, elixirs, or microcapsules, or parenterally in the form of injections of sterile solutions or suspensions with water or any other pharmaceutically acceptable liquid as needed. The compounds may be formulated by adequately combining them with pharmacologically acceptable carriers or media, specifically, sterilized water or physiological saline, vegetable oil, emulsifiers, suspensions, surfactants, stabilizers, flavoring agents, excipients, vehicles, preservatives and bonding agents, and mixing in a unit dose form required for generally accepted drug implementation. The amount of each effective ingredient in these preparations is provided for giving a suitable dosage acquirable within the indicated range.
Additives mixable in the tablets and capsules include a bonding agent such as gelatin, corn starch, tragacanth gum or arabic gum; an excipient such as crystalline cellulose; a swelling agent such as corn starch, gelatin or alginic acid; a lubricator such as magnesium stearate; a sweetener such as sucrose, lactose or saccharin; and a flavoring agent such as peppermint, Gaultheria adenothrix oil and cherry. When the unit dosage form is a capsule, a liquid carrier such as oil can also be included in the above ingredients. Sterile compositions for injections can be formulated following normal drug implementations using vehicles such as distilled water used for injections.
Aqueous solutions for injection include physiological saline, and isotonic liquids containing glucose or other adjuvants, such as D-sorbitol, D-mannose, D-mannitol, and sodium chloride. These solutions can be used in conjunction with suitable dissolution adjuvants, such as alcohol, specifically ethanol, polyalcohols such as propylene glycol and polyethylene glycol, and non-ionic surfactants such as poly sorbate 80 (TM) and HCO-50. Sesame oil or Soy-bean oil can be used as an oleaginous liquid and may be used in conjunction with benzyl benzoate or benzyl alcohol as dissolution adjuvants. They may also be formulated with a buffer, such as phosphate and sodium acetate buffer; a pain-killer, such as procaine hydrochloride; a stabilizer such as benzyl alcohol, phenol; and an anti-oxidant. The injection solution thus prepared is filled into a suitable ampoule.
Administration to the patients may be carried out using a method well known to one skilled in the art, for example, via intranasal, bronchial, intramuscular or oral route, as well as intra-arterial, intravenous or subcutaneous injection. While the doses vary depending on the patient""s body weight and age and on the administration method, one skilled in the art may properly determine the adequate doses. If the compound can be encoded by DNA, gene therapy may possibly be carried out by incorporating the DNA into a vector for gene therapy. While the doses of the drug and the method for administration may vary depending on the patient""s body weight, age and condition, one skilled in the art may determine them properly. The administered dose of a compound that binds to the protein of the present invention or promotes or inhibits the activity of the protein of the present invention will vary depending on the patient""s condition. For oral administration, about 0.1 to about 100 mg per day, preferably about 1.0 to 50 mg per day, and more preferably about 1.0 to 20 mg per day may typically be administered to a normal adult (weighing 60 kg).
The parenteral dose to be administered varies depending on the subject for administration, target organ, subject""s conditions, and the method of administration. In the form of an injection, for example, a dose of about 0.01 to about 30 mg per day, preferably about 0.1 to about 20 mg per day, and more preferably about 0.1 to about 10 mg per day may be advantageously administered to a normal adult (weighing 60 kg) by intravenous injection. For other animals, the dose calculated to body weight of 60 kg may be administered to the animal.